Continued Development of Techniques for In Vivo Investigation of the Function of Identified Neurons One of our major aims is to understand how circuitry within the basal ganglia contributes to the control and learning of actions. An important component of this line of research is gaining an understanding of the activity of specific neuronal subtypes within this circuitry during the learning, planning, initiation and performance of actions and action sequences. We have worked with Drs. Rui Costa of the Champalimaud Neuroscience Institute and Steven Vogel of the Laboratory of Molecular Physiology at NIAAA to develop a technique for in vivo fiber photometry to perform Time Correlated Single Photon Counting (TCSPC) fluorimetry in the striatum in vivo. Coupling this technique with selective expression of a fluorescent calcium indicator protein (GCaMP) in either direct or indirect pathway striatal medium spiny projection neurons has allowed us to measure calcium transients reflective of neuronal activation. We found that neurons in the two pathways were co-active during initiation of movement sequences, particularly when the animal was moving away from the hemisphere in which the fiber probe was implanted. We are now extending this technique to measure intracellular calcium signals in different neuronal subtypes in different deep brain regions. We are also exploring the possibility of measuring calcium transients in presynaptic elements in awake, freely-moving mice using a new single-fiber chronically-implantable photometry system that we recently developed. We are interested in measuring intracellular signaling molecules other than calcium, in order to assess the dynamics of these signaling pathways during behavioral execution. Genetically-encoded detectors based on Forster Resonance Energy Transfer (FRET) have been developed to measure a variety of intracellular signaling molecules. The TCSPC capability of our fiber photometry system should allow us to measure changes in FRET using fluorescence lifetime changes in the donor flourophore of these molecules. Experiments in cell lines indicate that our system has the sensitivity to detect such changes, and we are currently testing some detectors in vivo. Ultimately, these experiments should allow us to measure a variety of intracellular signals in identified neurons within the corticostriatal circuitry. Cannabinoid 1 Receptors on Orbitofrontal-Striatal Synapses Control the Balance between Goal-Directed and Habitual Behavior Our previous studies indicated that endocannabinoids and CB1 cannabinoid receptors are involved in both short- and long-lasting depression of synaptic transmission at corticostriatal synapses. Furthermore, studies in the laboratory have shown that CB1 receptors play a crucial role in instrumental habit learning, as measured in a food-rewarded lever-pressing task. However, these receptors are expressed on presynaptic axon terminals in many locations throughout the cortico-basal ganglia circuitry where they could participate in such behaviors. The orbitofrontal cortex (OFC) projects to the dorsomedial striatum (DMS), and has important roles in maintaining goal-directed instrumental lever-pressing, presumably by providing information about outcome value. Thus, we reasoned that CB1-mediated depression at OFC to DMS synapses may aid in the development of habitual responding by dampening this outcome-related signal. To test this hypoethsis, we sought to selectively knock out CB1 receptors in this pathway and examine effects on instrumental learning and performance. We used mice carrying a floxed allele of the CB1 receptor to allow for conditional knockout of the protein in cells where the Cre recombinase is active. We initially used three approaches to selectively knock out CB1 in the OFC. The first strategy used a tamoxifen-inducible Cre-expressing mouse bred with the floxed-CB1 mouse, and tamoxifen injection into OFC. We also injected into the OFC a virus in which Cre expression is driven by a promoter that works well in most, if not all, neurons, as well as a virus containing a CamKIIalpha promoter to drive expression in OFC projection neurons. Under all three of these conditions, mice showed goal-directed responding in the instrumental task (e.g. they were deficient in habit learning). However, OFC projection neurons innervate several brain regions. To determine the role of CB1 in neurons projecting to the DMS, we used a dual-virus injection strategy in which a Cre recombinase construct that confers flipase-dependent expression was infected in OFC and a virus containing flipase was expressed in DMS of CB1-floxed mice. With this intersectional strategy we again observed impaired habit learning in mice, implicating CB1 in OFC-DMS projection neurons in transition from goal-directed to habit learning. All of the mice used in these experiments were otherwise healthy and showed normal lever-pressing rates, reward retrieval and consumption in the instrumental task. Overall, our findings support the idea that endocannabinoid/CB1-mediated suppression of OFC-DMS glutamatergic transmission has important roles in transition from goal-directed to habitual action control, presumably through suppression of information about outcome value.